Microbiological hydroxylation of aromatic acids

ABSTRACT

A benzoic acid having from zero to three methyl and/or halo substituents and having at least two adjacent unsubstituted ring carbon atoms can be converted to the corresponding dihydroxybenzoic acid derivative by the action of various species of Nocardia which are normally incapable of performing such a conversion. This is accomplished by first contacting the Nocardia cells with an aromatic or halogenated aromatic hydrocarbon for a time sufficient to induce the enzyme system of said cells to perform such an hydroxylation, and thereafter contacting the induced cells with the benzoic acid substrate under aerobic conditions.

United States Patent [151 3,645,847

EltZ Feb. 29, 1972 [54] MICROBIOLOGICAL HYDROXYLATION OF AROMATIC AClDSInventor: Robert W. Eltz, Media, Pa.

Assignee: Sun Oil Company, Philadephia, Pa.

Filed: Feb. 8, 1968 Appl. No.: 703,870

US. Cl ..195/30 Int. Cl. ..Cl2b l/00 Field ofSearch.... ...l95/28,30l,3l-l,5l

References Cited UNITED STATES PATENTS 3,383,289 5/1968 Raymond et al..195/28 l-losler ..195/28 Primary Examiner-A. Louis Monacell AssistantExaminer-Gary M. Nath Attorney-George L. Church, Donald R. Johnson,Wilmer E. McCorquodale, Jr. and Stanford M. Back ABSTRACT A benzoic acidhaving from zero to three methyl and/or halo substituents and having atleast two adjacent unsubstituted ring carbon atoms can be converted tothe corresponding dihydroxyben'zoic acid derivative by the action ofvarious species of Nocardia which are normally incapable of performingsuch a conversion. This is accomplished by first contacting the Nocardiacells with an aromatic or halogenated aromatic hydrocarbon for a timesufficient to induce the enzyme system of said cells to perform such anhydroxylation, and thereafter contacting the induced cells with thebenzoic acid substrate under aerobic conditions.

5 Claims, No Drawings MICROBIOLOGICAL IIYDROXYLATION OF AROMATIC ACIDSBACKGROUND OF THE INVENTION This invention relates to themicrobiological hydroxylation of unsubstituted and substituted benzoicacids having at least two adjacent unsubstituted ring carbon atoms bythe action of an induced micro-organism of the genus Nocardia which isnormally incapable of effecting such a conversion.

In U.S. Pat. Ser. No. 3,383,289, there is described a process for themicrobiological oxidation of methyl-substituted benzene hydrocarbons byvarious species of Nocardia to form, inter alia, a mixture of thecorresponding nonhydroxylated benzoic acids and the dihydroxylatedbenzoic acid analogues thereof. Of these two elaboration products, thedihydroxylated compound is commercially the more valuable of the twomaterials. Nevertheless, in each instance where the dihydroxylatedcompound was formed, substantial amounts of the nonhydroxylated analoguewere also recovered. Efforts to convert these nonhydroxylated byproductsto the more valuable dihydroxylated acids by microbial means hasheretofore proved to be entirely unsuccessful since, surprisingly, thespecies of Nocardia which were capable of producing a mixture of both ofthese products from methyl-benzenes appeared incapable of effecting thedesired conversion starting with the corresponding benzoic acid.

It is, therefore, an object of this invention to provide a novel processfor the preparation of dihydroxybenzoic acids from unsubstituted ormethyl and/or halogen substituted benzoic acids having at least twoadjacent unsubstituted ring carbon atoms. It is a further object of thisinvention to provide a process whereby such benzoic acids may beconverted to the corresponding dihydroxy analogues by subjecting them tothe 7 action of various species of Nocardia which are normally incapableof effecting such a conversion.

SUMMARY OF THE INVENTION In accordance with the present invention it hasnow been found that benzoicacids having from zero to three methyl and/orhalogen substituents and having at least two adjacent unsubstituted ringcarbon atoms may be dihydroxylated to form the correspondingdihydroxybenzoic acid derivative by subjecting said benzoic acidcompound under fermentation conditions to the microbiological activityof a species of Nocardia which is normally incapable of performing sucha conversion, said species of Nocardia having been previously contactedwith an aromatic or halogenated aromatic inducer defined hereinbelow fora time sufficient to induce the enzyme system of said micro-organism toeffect such a dihydroxylation.

PREFERRED EMBODIMENTS OF THE INVENTION The process of this invention maybe conveniently carried out by first establishing a growth of Nocardiacells in a nutrient medium, said cells being normally incapable ofhydroxylating a benzoic acid but whose enzyme system is capable of beingso induced; thereafter inducing said cells by adding to a dispersion ofthem an hydroxylation-inducing amount of an inducer as defined belowunder growth conditions for a period sufficient to induce the cellsenzyme systems to dihydroxylate the above-defined benzoic acid;thereafter subjecting said benzoic acid to the action of the inducedcells in the presence of a nutrient medium and under aerobicfermentation conditions; and recovering from the fermentation broth adihydroxylated derivative of said benzoic acid. Optionally, the benzoicacid substrate may be added to the fermentation medium at the same timethat the inducer is added. Under these conditions, however, an inductionperiod of several hours must pass before production of thedihydroxylated product is observed.

In preparing a species of Nocardia for use in the present process, aculture sample is desirably transferred from an agar slant to a shakeflask containing a nutritionally adequate medium, including a carbonsource on which the organism can grow. In some cases it may be desirablealso to have an additional growth-stimulating material present such aspeptone, beef extract, yeast extract or the like, although the additionof such materials is not essential. The mixture is then incubated atabout 30 C. Either immediately, or after an incubation period of up toabout 24 hours depending on the concentration of cells, the inducer isadded to the growing culture.

The nutrient medium into which the selected organism is inoculated mustcontain as mentioned above, in addition to inorganic nutrient and asource of nitrogen, a carbon source which also provides the organismwith its energy. While in general any organic substance containing acombined source of carbon and hydrogen, can be used, as for example,carbohydrates or fatty acids, it is preferred that hydrocarbons, andparticularly n-paraffins having from two to 30 carbon atoms, such asn-butane, n-dodecane, or n-hexadecane, be employed, and especially thelatter compound; Alternatively, depending upon the strain of organismwhich is selected, certain aromatic hydrocarbons such as benzene may beutilized instead. The carbon source, as for example n-hexadecane, may beadded in'one batch just prior'to inoculation of the medium. Preferably,however, it should be added periodically in small increments throughoutthe entire course of the fermentation in order to avoid concentrationsof this material in the medium which would be toxic to the organism.Although the amount to be added at any given time may vary, dependingupon the organism, it is generally desirably to have not more than about3 ml./liter of n-hexadecane in the vessel at any one time, for a totalof about 10 grams/liter of this carbon source, for any givenfermentation batch.

The source of nitrogen which is to be included in the nutrient mediumcan be any inorganic or organic nitrogencontaining compound which iscapable of providing nitrogen in a form suitable for metabolicutilization by the micro-organism such as proteins, amino acids,ammonium sulfate, ammonium phosphate, urea of the like.

The inorganic nutrient should be water soluble and provide an adequatesource of minerals, preferably in the form of their salts such as iron,sodium and phosphorus compounds. One example of a nutritionally adequatemedium, in addition to the carbon-energy source, is as follows:

Grams per liter of water urea 2.0 yeast extract (Difco) 0.5 M so,-7H,o0.2 Na.,CO 0.1 CaC l,-2H,0 0.01 MnSO,-H,0 0.02 Feso,-7Y,o 0.005 KH,PO,0.8 Nan-1P0, 1.2

both before and after the addition of the substrate, can be varied fromabout 20 to 45 C., and preferably is from 25 to 35 C. to ensure adequategrowth of the organism. The pH of the fermentation medium should bemaintained within a range of from 5.5 to 9.5, and preferably 7.0 to 8.5.Adjustments are often necessary during the course of the fermentation inorder to keep the pH within the preferred ranges. This is generally 7achieved by the periodic addition of sufficient amounts of an alkalimetal hydroxide. Following the initial introduction of the substrate tothe established fermentation medium, the period of fermentationconstituting the second stage of the process should continue for about24 to 84 hours, and

preferably for about 36 to 60 hours.

The compounds which have been found to induce the enzyme systems ofNocardiain order that they may convert benzoic acid compounds to theirdihydroxy counterparts are aromatics or halogenated aromatics having atleast one aromatic ring with not more than one halogen substituentattached thereto. Desirably, though not essentially, these inducercompounds should contain at least two adjacent ring carbon atoms whichare unsubstituted. Preferably, also these inducers should be nonvolatileand nonmetabolizable by the Nocardia organism, although this is notessential either.

Examples of compounds which have satisfactorily induced Nocardia cellsto hydroxylate benzoic acid compounds are hydrocarbons such as p-xylene,m-xylene, o-xylene, toluene, benzene, biphenyl, naphthalene,Z-methyl-naphthalene, and tetralin; and halogenated hydrocarbons such aspchlorotoluene or chlorobenzene. These compounds are desirably added tothe growing Nocardia cells prior to the addition of the benzoic acidsubstrate in hydroxylation-inducing amount, but in amounts which willnot be toxic to the microorganism. These amounts will vary dependingupon the inducer and the organism which is used, but should desirably bepresent in the fermentation medium in amounts greater than p.p.m. ofmedium, and preferably about 20-200 p.p.m. The inducer is preferablyadded in amounts sufficient to maintain these concentrations in themedium over a period of time until induction is complete, usually withinabout 1-12 hours, depending on the growth rate of the organism.

The benzoic acid substrates to be hydroxylated, are as described above,those compounds having from zero to three methyl and/or halosubstituents on the aromatic ring and which, additionally, have at leasttwo adjacent unsubstituted ring carbon atoms. Examples of benzoic acidsubstrates which may be used in this process are suchcompounds asbenzoic acid; p-toluic acid; o-toluic acid; m-toluic acid;2,5-dimethylbenzoic acid; 2,3-dimethyl-benzoic acid;2,3,4-trimethylbenzoic acid, 3-methyl-4-chlorobenzoic acid;p-chloro-benzoic acid and thelike. It will be understood, of course,that inasmuch as the medium must desirably be kept at a neutral pH orhigher, the acid substrates will generally be present in the reactionmedium in the form of their salts.

When the aforementioned compounds are subjected to the action of theinduced Nocardia cells, there are obtained the correspondingdihydroxylated benzoic acids, such as dihydroxy-benzoic acid;2,3-dihydroxy-p-toluic acid; 2,3- dihydroxy-4,6-dimethyl-benzoic acid;dihydroxy-5,6-dimethyl benzoic acid;2,3-dihydroxy-4-chloro-S-methylbenzoic acid;2,3-dihydroxy-p-chloro-benzoic acid; and 2,3dihydroxy-4,5,6-trimethyl-benzoic acid, generally in their alkali metal salt form.

The benzoic acid substrate is desirably added periodically to thefermentation medium in small increments in the form of its water-solublesalt throughout the total fermentation period following the inductionperiod. Together with the substrate should be added small amounts of theaforementioned carbon source, such as n-hexadecane. The amount of eachportion of substrate which is added to the medium is generally in therange of from 0.2 to 0.5 gram/liter, and preferably less than about 1gram/liter.

The dihydroxylated products are conveniently recovered from thefermentation medium by conventional means. Thus, for example, the cellsmay be separated from the broth by centrifugation or filtering, and theclear broth acidified and treated by extraction with a suitable solventsuch as ether, dioxane, or amyl acetate.

The species of Nocardia which may suitably be employed in the process ofthis invention are those which are normally incapable of converting abenzoic acid to its dihydroxy derivative, but whose enzyme systems arenevertheless capable of being so induced, as described above. Onepreferred organism for practising this invention is Nocardiasdlmonicolor, ATCC No. 19,149.

Other species of Nocardia which are likewise suitable are those taughtin copending Ser. No. 509,621, mentioned above, which describes severalspecies of Nocardia that are capable of oxidizing methylbenzenecompounds. Thus, there may advantageously be employed the followingadditional species of Nocardia, all of which have been classified byBergeys Manual and which further have been deposited with the AmericanType Culture Collection, Washington, D.C.:

l. A wild-type strain obtained from soil in Alabama, havingcharacteristics approximately those set forth for Nocardia corallina inBergeys Manual and hence classified as such species. A culture of thisstrain has been deposited with the American Type Culture Collection inWashington, D.C., under the number ATCC 19,070. Colonies of thismicro-organism have an orange color.

2. A reddish-colored mutant obtained by ultraviolet irradiation of ATCCNo. 19,070. The mutant has also been deposited with the American TypeCulture Collection and has been designated as ATCC No. 19,071.

3. A strain isolated from Pennsylvania soil and likewise classified asNocardia corallina. This micro-organism is orange colored like thefirst-mentioned wild-type specimen but shows distinct differences inenzymaticoxidative characteristics. A culture deposit of this strain hasbeen designated as ATCC No. 19,148.

4. Another soil isolate classified as per Bergeys Manual as Nocardiaminima and designated as ATCC No. 19,150.

It will be understood that in addition to the above species thisinvention also contemplates the use of other species of Nocardia whichmay routinely be identified by those skilled in the art in accordancewith the techniques taught herein to determine their applicability tothe present unique process. Amongst such species should be included theconstitutive mutants of the Nocardia micro-organisms defined above. Bythe term constitutive mutant" is meant those selected organisms whichhave been mutated by conventional means in order to convert them from aspecies which will not nonnally effect the desired conversion in theabsence of an inducer to a species which will thereafter perform such aconversion without an inducer having to be present. Examples of othertypes of constitutive mutants and methods for obtaining them are wellknown in the art; see, for example, Biochim. Biophys. Acta, (1964)609-610.

The following examples specifically illustrate embodiments of theinvention.

EXAMPLE 1 Grams per liter of water urea 2.0 yeast extract (Difco) 0.5mg. SO,-7H,O 0.2 Na,C0, 0.1 CaC1,21-1,0 0.01 MnS0,-H,O 0.02 Fe S0 7H,00.005 KH,P0 0.8 Na,HP0 1.2

The fermenter was a bafiled stainless steel tank of 12-inch innerdiameter, equipped with a 6-inch bladed turbine impeller. The fermenterwas operated at 30 C., with sterile air supplied at 0.33volume/volume/minute and an agitator speed 7 menter was supplied withn-hexadecane continuously at 5 ml./hour/tank from the start to the sixthhour and at 10 mL/hour/tank during the sixth to 17th hour period. At the17th hour, when moderate growth was reached, the pH of the culture wasadjusted to 8.0 with 8 percent aqueous NaOl-l additions as required.Intermittent benzene addition was also started at the 17th hour at sucha rate as to maintain the apparent benzene level in the culture withinthe range of 75-125 mg./liter, as determined by quantitative ultravioletanalysis of iso-octane extracts of the culture. Also at the 17th hour,20 grams of p-toluic acid (plus sufficient NaOl-l to neutralize the acidand dissolve it in water in the form of its sodium salt) was added tothe culture. Similar additions were made at the 42nd, 53rd, and 62ndhours, to maintain the p-toluic acid concentration within the 0.3-0.5gram/liter range. Colorimetric assay for 2,3-dihydroxy-p-toluic acid(DHPT) showed 0.89 gram/liter at the 62nd hour. and 1.0 gram/liter atthe 70th hour. The presence of DHPT was also confirmed by ultravioletspectrum and typical mobility and reaction on paper chromatograms. Thep-toluic acid was seen to disappear concurrently with DHPT accumulation,as observed on paper chromatograms. Trials made in a manner similar tothis fermenter run, but omitting the benzene, showed no disappearance ofp-toluic acid or formation of DHPT.

EXAMPLE 2 A fermenter containing 32 liters of sterile medium wasprepared and inoculated as described in example I. The fermenter wasoperated at 30 C., with sterile air supplied at 0.20volume/volume/minute and an agitator speed of 500 r.p.m.Normal-hexadecane was supplied continuously at ml./hour/tank for thefirst 9 hours after inoculation. Ten grams of biphenyl dissolved in 40ml. of n-hexadecane was added continuously between the 19th and 22ndhours; a similar addition was made between the 57th and 59th hours. Atthe 20th hour, the pH of the culture was adjusted up to 8.0 with 8percent aqueous NaOl-l solution and maintained at that pH throughout therun with NaOH additions as required. Between the 22nd and 45th hours, a20 percent (w./v.) aqueous solution of p-toluic acid (plus sufficientNaOl-l to neutralize the acid and dissolve it in water in the form ofits sodium salt) was added at the rate of 26 ml./hour/tank. The brothsupematant fluids, as assayed by quantitative ultraviolet analysis,contained 1.25 grams/liter DHPT after 45 hours and 2.0 grams/liter after66 hours. The presence of DHPT was confirmed by typical paperchromatographic mobility and color reactions. Control runs of thisnature from which biphenyl was omitted, showed no p-toluic acidutilization or DHPT formatron.

EXAMPLE 3 A growing culture of Nocardia was prepared in the mannerdescribed for inoculation preparation in example l. A 35-ml. portion wastransferred to a 300-ml. culture flask, to which was added 4.5 ml. ofwater, 0.5 ml. of an aqueous sodium ptoluate solution (8 percent w./v.in free acid) and 0.05 ml. of a 30 percent (v./v.) tetralin solution inn-hexadecane. After shaking the flask culture at 30 C. for 9 hours, 0.05gram/liter of Dl-lPT had accumulated in the broth. Parallel flasks, fromwhich sodium p-toluate or tetralin were omitted, showed no DHPT present.

EXAMPLE 4 A growing culture of Nocardia was grown and exposed to pxylenein a ferrnenter in the same manner as the culture was grown and exposedto benzene in example l, but without ptoluate added. After 7 hours ofp-xylene exposure, a 240-ml. sample was removed from the fermenter, thecells were removed from the broth by centrifugation, washed once byresuspending them in 240 ml. of carbon-free mineral salts medium,recentrifuged, and finally resuspended in 240 ml. of the same medium.Thirty-five milliliters of this cell suspension were placed in a cultureflask to which was added 0.5 ml. of aqueous sodium p-toluate solution (8percent w./v. in free acid). After shaking the flask culture at 30 C.for 1 hour, 0.15

gram/liter DHPT was observed in the broth. A similar flask from whichthe sodium p-toluate was omitted showed no DHPT. In addition, cellsprepared in a similar way but with omission of p-xylene addition fromthe fermenter growth stage, showed no ability to convert sodiump-toluate to DHPT when assayed in washed cell flask experiments in thissame manner as the p-xylene-exposed cells.

EXAMPLE 5 The procedures of example 3 were repeated except that mxylenewas substituted for tetralin and the flask was immediately stopped witha cork stopper. After 22 hours, 0.02 gram/liter of DHPT were observed inthe broth. The control flask containing no rn-xylene did not produce anyDHPT whatever.

EXAMPLE 6 The procedures of example 3 are repeated except that oxyleneis substituted for tetralin and the flask is stopped immediately with acork stopper. The organism employed is Nocardia corallina ATCC No.19,148. After 22 hours, 0.04 gram/liter of DHPT is observed. No DHPT isfound in the control flask.

EXAMPLE 7 The procedures of example 3 were repeated substituting 2-methylnaphthalene for tetralin. After 22 hours, 0.11

, gram/liter of DHPT were observed in the broth while no DHPT wasobserved in the control flask.

EXAMPLE 8 The procedures of example 3 were repeated substitutingnaphthalene for tetralin. After 22 hours, 0.11 gram/liter of DHP'T wereobserved in the broth while no DHPT was observed in the control flask.

EXAMPLE 9 EXAMPLE 10 The procedures of example 4 are repeatedsubstituting pchlorobenzoic acid in the washed-cell flask experiments.The organism employed is Nocardia sp., ATCC No. 19,070. After 1 hour ofshaking at 30 C., 0.08 gram/liter of 2,3-dihydroxy-4- chlorobenzoic acidare observed.

EXAMPLE 11 The procedures of example 4 are repeated substituting toluenefor p-xylene in the fermenter growth tank. The organism employed isNocardia sp. ATCC No. 19,071. After 22 hours, 0.04 gram/liter of DHPTare observed.

EXAMPLE 12 The procedures of example 3 were repeated substitutingmesitylene for tetralin. The results were negative and no DHPT whateverwas observed.

EXAMPLE 13 The procedures of example 3 were repeated substituting durenefor tetralin. The results were negative and no Dl-IPI whatever wasobserved.

EXAMPLE 14 The procedures of example 3 were repeated substitutingpdichlorobenzene for tetralin. The results were negative and no DHPTwhatever was observed.

The foregoing examples specifically illustrate the preparation of2,3-dihydroxy-p-toluic acid starting with p-toluic acid. However,fermentations with other benzoic acids enumerated above give analogousresults. This is true regardless of the inducer which is employed.

The dihydroxy benzoic acids prepared by the present invention arevaluable products having various applications of commercial interest,and particularly as chelating agents, metal deactivators, and dyeintermediates.

What is claimed is:

l. A method of hydroxylating a benzoic acid having zero to threesubstituents of the class consisting of methyl I and halogen groups andhaving at least two adjacent unsubstituted ring carbon atoms to form thecorresponding dihydroxylated benzoic acid derivative which comprises:

a. establishing a growth of micro-organism cells in a nutrient medium,said micro-organism being a Nocardia species normally incapable ofhydroxylating said benzoic acid but being capable of induction-f suchactivity;

b. inducing said species by incorporating into said growth ofmicro-organism cells in a nutrient medium an hydroxylation-inducingamount of a compound selected from the group consisting of p-xylene,m-xylene, o-riylene, benzene, toluene, biphenyl, naphthalene,2-rriethylnaphthalene, tetralin, and p-chlorotoluene, and contacting thecells therewith under aerobic conditions;

c. thereafter subjecting said benzoic acid to the action of the inducedcells under aerobic fermentation conditions;

d. and recovering from the fermentation medium a dihydroxylated productof said benzoic acid.

2. The process according to claim 1 wherein said fermentation conditionsinclude a pH level of from about 5.5 to 9.5.

3. The process according to claim 1 wherein the induced species ofNocardia is a member of the species Nocardia coral- Iina, Nocardiasalmonicolor, or Nocardia minima.

4. The process according to claim 1 wherein the micro-organism isselected from the group consisting of ATCC No. 19,070; ATCC No. 19,071;ATCC No. 19,148; ATCC No. 19,149; and 19,150.

5. The process according to claim 1 wherein the benzoic acid is p-toluicacid, the inducer is biphenyl, the induced organism is Nocardiasalmonicolar, ATCC No. l9,l49, and the product recovered is2,3-dihydroxy-p-toluic acid.

2. The process according to claim 1 wherein said fermentation conditionsinclude a pH level of from about 5.5 to 9.5.
 3. The process according toclaim 1 wherein the induced species of Nocardia is a member of thespecies Nocardia corallina, Nocardia salmonicolor, or Nocardia minima.4. The process according to claim 1 wherein the micro-organism isselected from the group consisting of ATCC No. 19,070; ATCC No. 19,071;ATCC No. 19,148; ATCC No. 19,149; and 19,150.
 5. The process accordingto claim 1 wherein the benzoic acid is p-toluic acid, the inducer isbiphenyl, the induced organism is Nocardia salmonicolor, ATCC No.19,149, and the product recovered is 2,3-dihydroxy-p-toluic acid.